Lawrence A. Lacey

Insect pathogens as biological control agents: Back to the future.

The development and use of entomopathogens as classical, conservation and augmentative biological control agents have included a number of successes and some setbacks in the past 1years. In this forum paper we present current information on development, use and future directions of insect-specific viruses, bacteria, fungi and nematodes as components of integrated pest management strategies for control of arthropod pests of crops, forests, urban habitats, and insects of medical and veterinary importance. Insect pathogenic viruses are a fruitful source of microbial control agents (MCAs), particularly for the control of lepidopteran pests. Most research is focused on the baculoviruses, important pathogens of some globally important pests for which control has become difficult due to either pesticide resistance or pressure to reduce pesticide residues. Baculoviruses are accepted as safe, readily mass produced, highly pathogenic and easily formulated and applied control agents. New baculovirus products are appearing in many countries and gaining an increased market share. However, the absence of a practical in vitro mass production system, generally higher production costs, limited post application persistence, slow rate of kill and high host specificity currently contribute to restricted use in pest control. Overcoming these limitations are key research areas for which progress could open up use of insect viruses to much larger markets. A small number of entomopathogenic bacteria have been commercially developed for control of insect pests. These include several Bacillus thuringiensis sub-species, Lysinibacillus (Bacillus) sphaericus, Paenibacillus spp. and Serratia entomophila. B. thuringiensis sub-species kurstaki is the most widely used for control of pest insects of crops and forests, and B. thuringiensis sub-species israelensis and L. sphaericus are the primary pathogens used for control of medically important pests including dipteran vectors. These pathogens combine the advantages of chemical pesticides and MCAs: they are fast acting, easy to produce at a relatively low cost, easy to formulate, have a long shelf life and allow delivery using conventional application equipment and systemics (i.e. in transgenic plants). Unlike broad spectrum chemical pesticides, B. thuringiensis toxins are selective and negative environmental impact is very limited. Of the several commercially produced MCAs, B. thuringiensis (Bt) has more than 50% of market share. Extensive research, particularly on the molecular mode of action of Bt toxins, has been conducted over the past two decades. The Bt genes used in insect-resistant transgenic crops belong to the Cry and vegetative insecticidal protein families of toxins. Bt has been highly efficacious in pest management of corn and cotton, drastically reducing the amount of broad spectrum chemical insecticides used while being safe for consumers and non-target organisms. Despite successes, the adoption of Bt crops has not been without controversy. Although there is a lack of scientific evidence regarding their detrimental effects, this controversy has created the widespread perception in some quarters that Bt crops are dangerous for the environment. In addition to discovery of more efficacious isolates and toxins, an increase in the use of Bt products and transgenes will rely on innovations in formulation, better delivery systems and ultimately, wider public acceptance of transgenic plants expressing insect-specific Bt toxins. Fungi are ubiquitous natural entomopathogens that often cause epizootics in host insects and possess many desirable traits that favor their development as MCAs. Presently, commercialized microbial pesticides based on entomopathogenic fungi largely occupy niche markets. A variety of molecular tools and technologies have recently allowed reclassification of numerous species based on phylogeny, as well as matching anamorphs (asexual forms) and teleomorphs (sexual forms) of several entomopathogenic taxa in the Phylum Ascomycota. Although these fungi have been traditionally regarded exclusively as pathogens of arthropods, recent studies have demonstrated that they occupy a great diversity of ecological niches. Entomopathogenic fungi are now known to be plant endophytes, plant disease antagonists, rhizosphere colonizers, and plant growth promoters. These newly understood attributes provide possibilities to use fungi in multiple roles. In addition to arthropod pest control, some fungal species could simultaneously suppress plant pathogens and plant parasitic nematodes as well as promote plant growth. A greater understanding of fungal ecology is needed to define their roles in nature and evaluate their limitations in biological control. More efficient mass production, formulation and delivery systems must be devised to supply an ever increasing market. More testing under field conditions is required to identify effects of biotic and abiotic factors on efficacy and persistence. Lastly, greater attention must be paid to their use within integrated pest management programs; in particular, strategies that incorporate fungi in combination with arthropod predators and parasitoids need to be defined to ensure compatibility and maximize efficacy. Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are potent MCAs. Substantial progress in research and application of EPNs has been made in the past decade. The number of target pests shown to be susceptible to EPNs has continued to increase. Advancements in this regard primarily have been made in soil habitats where EPNs are shielded from environmental extremes, but progress has also been made in use of nematodes in above-ground habitats owing to the development of improved protective formulations. Progress has also resulted from advancements in nematode production technology using both in vivo and in vitro systems; novel application methods such as distribution of infected host cadavers; and nematode strain improvement via enhancement and stabilization of beneficial traits. Innovative research has also yielded insights into the fundamentals of EPN biology including major advances in genomics, nematode-bacterial symbiont interactions, ecological relationships, and foraging behavior. Additional research is needed to leverage these basic findings toward direct improvements in microbial control.

Field Manual of Techniques in Invertebrate Pathology

Bateman RP, Matthews GA, Hall F, 2007, Ground-based Application Equipment., Field Manual of Techniques in Invertebrate Pathology, Editors: Lacey. The Manual of Biocontrol Agents 4th Ed. British Crop Production (2007) Field Manual of Techniques in Invertebrate Pathology 2nd edition. Kluwer Academic. Geological Field Manual on ResearchGate, the professional network for scientists. Field Manual of Techniques in Invertebrate Pathology, Second Edition Field. The Manual of Biocontrol Agents (2009: formerly the Biopesticide Manual) gives a (2007) Field Manual of Techniques in Invertebrate Pathology 2nd edition. Insect pathology and fungal endophytes. FE Vega Insect pathology. Academic Press Field manual of techniques in invertebrate pathology, 153-177, 2000.

Variability in susceptibility to simulated sunlight of conidia among isolates of entomopathogenic Hyphomycetes

The influence of simulated sunlight on survival of conidia of 4 species of entomopathogenic Hyphomycetes was investigated. Conidia from 65 isolates ofBeauveria bassiana, 23 ofMetarhizium anisopliae, 14 ofMetarhizium flavoviride and 33 isolates ofPaecilomyces fumosoroseus were irradiated by artificial sunlight (295 to 1,100 nm at an ultraviolet-B irradiance of 0.3 W m−2) for 0, 1, 2, 4 and 8 h. Survival was estimated by comparing the number of colony forming units (CFU) produced by conidia exposed to irradiation to the number of CFUs produced by an unexposed control. Survival decreased with increased exposure to simulated sunlight; exposure for 2 h or more was detrimental to all isolates tested. Overall, isolates ofM. flavoviride were the most resistant to irradiation followed byB. bassiana andM. anisopliae. Conidia ofP. fumosoroseus were most susceptible. In addition to the large interspecies differences in susceptibility to irradiation, there was also an intraspecies variation indicating that strain selection to irradiation tolerance may be important in the development of microbial control agents where increased persistence in an insolated environment is desirable.

Liquid culture production of desiccation tolerant blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus

Liquid media with differing carbon concentrations and carbon-to-nitrogen ratios were tested for production of desiccation tolerant blastospores of Paecilomyces fumosoroseus. While all media tested supported sporulation in submerged culture, high blastospore concentrations (5·8 × 108) spores ml−) were produced in media containing 80 g glucose l− and 13·2 g Casamino acids l− (MS medium) and a significantly higher percentage (79%) of these blastospores survived air drying. Media containing glucose concentrations greater than 20 g l − and Casamino acid concentrations between 13·2 and 40 g l− supported maximal production of desiccation tolerant blastospores. All 23 isolates of P. fumosoroseus grown in MS media produced high concentrations of desiccation tolerant blastospores. When stored at 4 °C, more than 60% of the lyophilized blastospores produced in MS medium were still viable after 7 months storage while less than 25% of the air-dried blastospores survived after 90 d storage. Standard whitefly bioassays were performed to compare air-dried blastospores of P. fumosoroseus ARSEF 4491 with solid substrate-produced conidia of Beauveria bassiana ARSEF 252. Air-dried blastospores of P. fumosoroseus gave LD50s of 60 and 113 blastospores mm− for the silverleaf whitefly (Bemisia argentifolii) in two separate bioassays with potency ratios (LD50 B. bassiana/LD50 P. fumosoroseus)of 3·9 and 3·8, respectively. These results have demonstrated that high concentrations of blastospores of P. fumosoroseus can be rapidly produced in liquid culture, remain viable following drying, and infect and kill silverleaf whitefly.

Chapter I – Initial handling and diagnosis of diseased insects

Publisher Summary This chapter provides general guidelines for the recognition, handling and initial diagnosis of diseased insects and the identification of major entomopathogen groups. Insects are associated with a broad diversity of microorganisms in a variety of symbiotic relationships including commensalism, mutualism, and parasitism. There is an astronomical number of entomopathogens which cause diseases and there is a great a number of insect hosts to find them. Both living and dead insects that are patently infected with entomopathogens can be found in virtually every setting inhabited by insects including natural terrestrial and aquatic ecosystems, agroecosystems and in laboratory and commercial insect colonies. The recognition of diseased insects in the field or subsequently in the lab will initially rely on gross pathology and patent infections. Insects that are patently infected with entomopathogens often manifest characteristic symptoms and signs of diseases. Color changes due to entomopathogens in living insects are usually associated with those insects with transparent to semitransparent integuments. It is suggested that when an insect is suspected of being infected with an entomopathogen, it should be examined as soon as possible after collection. The invasion of cadavers by fast-growing saprophytic organisms may complicate the determination of the true etiological agent. The general characteristics of insect disease caused by the major groups of entomopathogens are also elaborated.

Ovicidal and Larvicidal Activity of Conidia and Blastospores of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) Against Bemisia argentifolii (Homoptera: Aleyrodidae) with a Description of a Bioassay System Allowing Prolonged Survival of Control Insects

Fungi are the only effective entomopathogens of members of the Aleyrodidae and other homopterans because of the piercing and sucking feeding strategy of these insects. The laboratory assessment of the entomopathogenic activity of fungi often requires a prolonged period of observation. Leaf quality can be the limiting factor in bioassays of fungi against whiteflies that require longer observation periods or those that require conditions that are as close to natural ones as possible. A bioassay system is described that utilizes rooted cabbage leaves infested with the highly polyphagous whitefly, Bemisia argentifolii . Using this method, discriminating dosages of aerial conidia and blastospores of two isolates of Paecilomyces fumosoroseus (Pfr) were bioassayed against eggs and nymphs of B. argentifolii . Low, but significant, mortality (10-20%) of eggs was observed 14 days after exposure to 3.8 104 blastospores/cm2 for the Pfr 97 and European Biological Control Laboratory (EBCL) Pfr 42 isolates. Additionally...

Dissemination of beneficial microbial agents by insects

Recent trends in pest management show an increased shift from insecticidal sprays towards the use of transgenic crops with insecticidal properties. This shift is partly due to the development of insect resistance to conventional insecticides and the large and increasing costs of developing and registering new chemicals. However, the long-term effectiveness of transgenic crops remains a hotly-debated issue. This scenario confronts and challenges entomologists to develop and evaluate other methods of pest management.

Pathogenesis and midgut histopathology of Bacillus thuringiensis in Simulium vittatum (Diptera: Simuliidae)

Abstract The pathogenesis and midgut histopathology which resulted when larvae of the blackfly, Simulium vittatum , were exposed to Bacillus thuringiensis at various temperatures and periods of exposure were investigated. The onset of mortality was studied at 10°, 15°, 19°, and 24°C. For each 4–5°C increase in temperature above 15°C, the onset of mortality was shortened by 24 hr. Exposures as brief as 15 min to 10 ppm of a whole spore preparation resulted in an average mortality of 29% in late-instar larvae. Mortality increased sharply for exposures up to 3 hr, approaching a maximum of 80%. The gross signs of disease included cessation of feeding and tetany with brachytosis. The tissue most affected was the midgut epithelium in the regions of the gastric caeca and posterior stomach. The formation of cytoplasmic vacuoles followed by cell lysis and/or sloughing were very apparent in moribund larvae. Death resulted without bacteremia.

Route of invasion and histopathology of Metarhizium anisopliae in Culex quinquefasciatus

Abstract The fungus Metarhizium anisopliae , known to be a pathogen of terrestrial insects, has been shown to be a potentially successful biocontrol agent of the Culicidae. Two methods of inoculation of Culex quinquefasciatus were examined and compared as to rate of kill, route of invasion, histopathology and pathogenesis, and presence or absence of toxin-related mortality. One method of inoculation involves covering the surface of the water containing the larvae with dry, hydrophobic conidia. Larvae contact the fungus when they break the water tension with their perispiracular valves for air intake. Histopathological studies reveal complete or near complete blockage of trachea with mycelial growth, beginning at the siphon tip and extending only one-third of the way into the trachea. Extensive hyphal growth and formation of appressoria with a subsequent melanization reaction by host tissue were clearly observed in the portion of the siphon invaded, but no hyphal bodies were evident, as occur in terrestrial insects. The waxy-coated conidia adhere to the inside surface of the valves, germinate, and invade the siphon tip tissue, much like the first stages of invasion seen in terrestrial insects. Death did not begin in the treated population until 48 hr post-treatment and appeared to be due primarily to suffocation. An alternate route of exposure examined is ingestion of detergent-wetted conidia that sink to the bottom of the container of water. When ingested, the conidia completely filled the larval gut, causing mortality to begin within 6 to 24 hr after ingestion. Toxin is apparently released during digestion; partially digested conidia are evident in electron micrographs of midguts of moribund larvae. Some conidia were shown to be at the begining stages of germination in the midguts of moribund larvae but no invasion of tissue was apparent. In both treatments, conidia must be viable in order to cause mortality in mosquito larvae.

An Attractant Trap for Autodissemination of Entomopathogenic Fungi into Populations of the Japanese Beetle Popillia japonica (Coleoptera: Scarabaeidae)

Autodissemination may be effective against the Japanese beetle, Popillia japonica Newman, in situations where habitats of its larvae are inaccessible. Trapping systems with attractants for both male and female Japanese beetles are commercially available. We fabricated an inoculation chamber which fits between the top of a standard Trece Catch CanTM Japanese beetle Trap and its holding canister. Beetles which are attracted to the trap fall through a hole in the inoculation chamber and land on a mesh screen. A partial funnel and canister attachment from a metal Ellisco Japanese Beetle Trap was secured beneath a hole in the floor at the opposite end of the chamber. A 10-cm section in the middle of the box, between the entrance hole in the roof and the exit hole in the floor, allows space for a dish containing the inoculum to be placed into the chamber through a door in the side of the unit. The trap has been tested with Metarhizium anisopliae (Metschnikoff) Sorokin as the pathogen. Beetles emerging from the ...